The present invention related to a method of determining triglycerides in biological fluids according to an enzymatic reaction and a reagent for such method.
There has been a need for the determination of serum triglycerides ever since there has been evidence indicating a relationship between fat metabolism and such disorders as atherosclerosis, diabetes, hypertension, and increased risk of myocardial infarction.
The first direct procedure for the determination of triglycerides was published in 1957 by E. Van Handel and D. B. Zilversmit (J. Lab. and Clin. Med. (1957) 50, No. 1, p. 152). This procedure uses organic solvents to extract triglyerides from serum and solid phase adsorption to remove interferring phospholipids. Extracted triglycerides are saponified with KOH releasing glycerol. Glycerol is oxidized with periodate to formaldehyde. This is condensed with chromotropic acid to form a color complex. This basic procedure has been modified, but, in all the modifications, the chemistry and method of this system have remained essentially unchanged.
In 1966, M. Eggstein (Klin. Woochenschr 44, (1966), pp. 262-266) introduced a partially enzymatic method for determining serum triglycerides. In Eggstein's method the glycerol released from saponified triglyceride is phosphorylated by adenosine triphosphate (ATP) in the presence of an ATP-regenerating system sustained by phosphoenol-pyruvate degrading to pyruvate. A subsequent reduction of pyruvate to lactate by reduced nicotinamide adenine dinucleotide (i.e., NADH) is followed spectrophotometrically and is directly related to the glycerol concentration.
In 1973, G. Bucolo and H. David (Clin. Chem. 19 (1973), pp. 476-482) introduced a totally enzymatic procedure for the determination of serum triglycerides. In their method, alkaline saponification is replaced by an enzymatic hydrolysis: EQU Triglycerides .sup.Lipase Glycerol+Fatty Acids (FFA)
The glycerol released by the enzymatic hydrolysis reacts according to the method presented by Eggstein. This method provided a clear improvement over the purely chemical and partial enzymatic methods, but it left much to be desired for the following reasons:
(a) the presence of internal blank rates; PA1 (b) the instability of reconstituted reagents; and PA1 (c) the necessity of rigid timing sequences. PA1 lipoprotein lipase (LPL) PA1 Porcine pancreatic lipase PA1 Rhizopus arrhizus lipase PA1 Candida cylindracea lipase PA1 Pseudomanas lipase
There have been attempts to overcome the disadvantages of the method of Bocolo and David. These include an enzymatic reaction as described in French Pat. No. 2,314,497. In this French patent, enzymatic methods are described for the determination of glycerol which has been hydrolyzed from triglycerides by the reaction of a solution of Rhizopus Delemar lipase and alpha-chrymotrypsin. The glycerol involves the reduction of NAD by glycerol and utilizes either a single enzyme, glycerol dehydrogenase (GDH), or two enzymes, glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH). In both methods, the reaction yields reduce nicotinamide adenine dinucleotide (i.e., NADH) which quantity is proportional to that of glycerol.
The principal objective of the French invention is to provide a method of glycerol determination which allows for a quantitative assay in visible light.
Thus, there still remains a need for a dependable, specific enzymatic reaction to determine triglycerides in biological fluids. This need is satisfied as described below by the present invention.